The genus Rickettsia, including Rickettsia prowazekii, the etiological agent of epidemic typhus, is unique, even amongst the intracellular bacterial parasites. These organisms grow directly in the cytoplasm, rather than in a vacuole in the cytoplasm of their eukaryotic host cells. We remain intensely interested in the biochemical and physiological mechanisms by which this organism copes with the problems and exploits the opportunities of this unique environmental niche and seek to define the mechanisms by which the rickettsiae enter the host cell cytoplasm. During the next five years we hope to answer key questions concerning 1) the transport systems for those nutrients that the rickettsia encounters in the cytoplasm, the metabolic capabilities of R. prowazekii for these substrates and the regulation of these processes; 2) the penetration mechanism used by rickettsiae to infect eukaryotic cells with emphasis on both the receptor interactions and the phospholipase A activity. The specific aims encompassed in these general areas are: I. Transport systems and metabolism: a) Development of the "rickettsiae with protein synthesis" as a system for transport studies of amino acids; b) Utilization of the corresponding "rickettsiae with m-RNA synthesis" to define the pathway for net ribonucleotide transport; c) Characterization of the ATP/ADP transport system in membrane vesicles using the cloned and expressed gene in E. coli; d) Investigation of growth and metabolism of rickettsiae growing in transport and respiratory-deficient somatic cell mutants; e) Characterization of the transport and metabolism of nucleotide sugars as a possible strategy for carbohydrate metabolism of nucleotide glycolytic enzymes; f) Investigation of proton translocation and its relationship to proton motive force in rickettsiae. II. Penetration-Receptors and Phospholipase A: a) Investigation of the role of phospholipase A2 activity in lysis of and entry into host cells; b) Characterization of receptors of the host-cell involved in rickettsia-host recognition using the Fluorescence Activated Cell Sorter.